RESUMEN
Inactivating mutations in human ecto-nucleotide pyrophosphatase/phosphodiesterase-1 (ENPP1) may result in early-onset osteoporosis (EOOP) in haploinsufficiency and autosomal recessive hypophosphatemic rickets (ARHR2) in homozygous deficiency. ARHR2 patients are frequently treated with phosphate supplementation to ameliorate the rachitic phenotype, but elevating plasma phosphorus concentrations in ARHR2 patients may increase the risk of ectopic calcification without increasing bone mass. To assess the risks and efficacy of conventional ARHR2 therapy, we performed comprehensive evaluations of ARHR2 patients at two academic medical centers and compared their skeletal and renal phenotypes with ENPP1-deficient Enpp1asj/asj mice on an acceleration diet containing high phosphate treated with recombinant murine Enpp1-Fc. ARHR2 patients treated with conventional therapy demonstrated improvements in rickets, but all adults and one adolescent analyzed continued to exhibit low bone mineral density (BMD). In addition, conventional therapy was associated with the development of medullary nephrocalcinosis in half of the treated patients. Similar to Enpp1asj/asj mice on normal chow and to patients with mono- and biallelic ENPP1 mutations, 5-week-old Enpp1asj/asj mice on the high-phosphate diet exhibited lower trabecular bone mass, reduced cortical bone mass, and greater bone fragility. Treating the Enpp1asj/asj mice with recombinant Enpp1-Fc protein between weeks 2 and 5 normalized trabecular bone mass, normalized or improved bone biomechanical properties, and prevented the development of nephrocalcinosis and renal failure. The data suggest that conventional ARHR2 therapy does not address low BMD inherent in ENPP1 deficiency, and that ENPP1 enzyme replacement may be effective for correcting low bone mass in ARHR2 patients without increasing the risk of nephrocalcinosis. © 2021 American Society for Bone and Mineral Research (ASBMR).
Asunto(s)
Terapia de Reemplazo Enzimático , Fosfatos , Adolescente , Animales , Suplementos Dietéticos , Humanos , Ratones , Fenotipo , Hidrolasas Diéster Fosfóricas/genética , PirofosfatasasRESUMEN
Enzyme replacement with ectonucleotide pyrophosphatase phospodiesterase-1 (ENPP1) eliminates mortality in a murine model of the lethal calcification disorder generalized arterial calcification of infancy. We used protein engineering, glycan optimization, and a novel biomanufacturing platform to enhance potency by using a three-prong strategy. First, we added new N-glycans to ENPP1; second, we optimized pH-dependent cellular recycling by protein engineering of the Fc neonatal receptor; finally, we used a two-step process to improve sialylation by first producing ENPP1-Fc in cells stably transfected with human α-2,6-sialyltransferase (ST6) and further enhanced terminal sialylation by supplementing production with 1,3,4-O-Bu3 ManNAc. These steps sequentially increased the half-life of the parent compound in rodents from 37 hours to ~ 67 hours with an added N-glycan, to ~ 96 hours with optimized pH-dependent Fc recycling, to ~ 204 hours when the therapeutic was produced in ST6-overexpressing cells with 1,3,4-O-Bu3 ManNAc supplementation. The alterations were demonstrated to increase drug potency by maintaining efficacious levels of plasma phosphoanhydride pyrophosphate in ENPP1-deficient mice when the optimized biologic was administered at a 10-fold lower mass dose less frequently than the parent compound-once every 10 days vs. 3 times a week. We believe these improvements represent a general strategy to rationally optimize protein therapeutics.
Asunto(s)
Antígenos de Histocompatibilidad Clase I/uso terapéutico , Hidrolasas Diéster Fosfóricas/farmacología , Ingeniería de Proteínas , Pirofosfatasas/farmacología , Receptores Fc/uso terapéutico , Proteínas Recombinantes de Fusión/farmacología , Calcificación Vascular/tratamiento farmacológico , Animales , Área Bajo la Curva , Modelos Animales de Enfermedad , Terapia de Reemplazo Enzimático/métodos , Glicosilación , Semivida , Antígenos de Histocompatibilidad Clase I/genética , Humanos , Masculino , Ratones Transgénicos , Hidrolasas Diéster Fosfóricas/genética , Hidrolasas Diéster Fosfóricas/aislamiento & purificación , Hidrolasas Diéster Fosfóricas/uso terapéutico , Estructura Terciaria de Proteína/genética , Pirofosfatasas/genética , Pirofosfatasas/aislamiento & purificación , Pirofosfatasas/uso terapéutico , Receptores Fc/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/uso terapéutico , Calcificación Vascular/genéticaRESUMEN
Ectonucleotide phosphatase/phosphodiesterase 1 (ENPP1) deficiency results in either lethal arterial calcifications ('Generalized Arterial Calcification of Infancy' - GACI), phosphate wasting rickets ('Autosomal Recessive Hypophosphatemic Rickets type 2' - ARHR2), early onset osteoporosis, or progressive spinal rigidity ('Ossification of the Posterior Longitudinal Ligament' - OPLL). As ENPP1 generates a strong endogenous mineralization inhibitor - extracellular pyrophosphate (PPi) - ENPP1 deficiency should not result in reduced bone volume, and therefore the mechanism ENPP1 associated osteoporosis is not apparent given current understanding of the enzyme's function. To investigate genetic pathways driving the skeletal phenotype of ENPP1 deficiency we compared gene expression in Enpp1asj/asj mice and WT sibling pairs by RNAseq and qPCR in whole bones, and in the liver and kidney by qPCR, directly correlating gene expression with measures of bone microarchitectural and biomechanical phenotypes. Unbiased analysis of the differentially expressed genes compared to relevant human disease phenotypes revealed that Enpp1asj/asj mice exhibit strong signatures of osteoporosis, ARHR2 and OPLL. We found that ENPP1 deficient mice exhibited reduced gene transcription of Wnt ligands in whole bone and increased transcription of soluble Wnt inhibitors in the liver and kidney, suggestive of multiorgan inhibition of Wnt activity. Consistent with Wnt suppression in bone, Collagen gene pathways in bone were significantly decreased and Fgf23 was significantly increased, all of which directly correlated with bone microarchitectural defects and fracture risk in Enpp1asj/asj mice. Moreover, the bone findings in 10-week old mice correlated with Enpp1 transcript counts but not plasma [PPi], suggesting that the skeletal phenotype at 10 weeks is driven by catalytically independent ENPP1 function. In contrast, the bone findings in 23-week Enpp1asj/asj mice strongly correlated with plasma PPi, suggesting that chronically low PPi drives the skeletal phenotype in older mice. Finally, correlation between Enpp1 and Fgf23 transcription suggested ENPP1 regulation of Fgf23, which we confirmed by dosing Enpp1asj/asj mice with soluble ENPP1-Fc and observing suppression of intact plasma FGF23 and ALP. In summary, our findings suggest that osteoporosis associated with ENPP1 deficiency involves the suppression of Wnt via catalytically independent Enpp1 pathways, and validates Enpp1asj/asj mice as tools to better understand OPLL and Paradoxical Mineralization Disorders.
Asunto(s)
Osteomalacia , Osteoporosis , Calcificación Vascular , Animales , Factor-23 de Crecimiento de Fibroblastos , Ratones , Osteoporosis/genética , Hidrolasas Diéster Fosfóricas/genética , Monoéster Fosfórico Hidrolasas , Pirofosfatasas/genéticaRESUMEN
Biallelic ENPP1 deficiency in humans induces generalized arterial calcification of infancy (GACI) and/or autosomal recessive hypophosphatemic rickets type 2 (ARHR2). The latter is characterized by markedly increased circulating FGF23 levels and renal phosphate wasting, but aberrant skeletal manifestations associated with heterozygous ENPP1 deficiency are unknown. Here, we report three adult men with early onset osteoporosis who presented with fractures in the thoracic spine and/or left radius, mildly elevated circulating FGF23, and hypophosphatemia. Total hip bone mineral density scans demonstrated osteoporosis (Z-score < -2.5) and HRpQCT demonstrated microarchitectural defects in trabecular and cortical bone. Next-generation sequencing revealed heterozygous loss-of-function mutations in ENPP1 previously observed as biallelic mutations in infants with GACI. In addition, we present bone mass and structure data as well as plasma pyrophosphate (PPi) data of two siblings suffering from ARHR2 in comparison to their heterozygous and wild-type family members indicative of an ENPP1 gene dose effect. The skeletal phenotype in murine Enpp1 deficiency yielded nearly identical findings. Ten-week-old male Enpp1 asj/asj mice exhibited mild elevations in plasma FGF23 and hypophosphatemia, and micro-CT analysis revealed microarchitectural defects in trabecular and cortical bone of similar magnitude to HRpQCT defects observed in humans. Histomorphometry revealed mild osteomalacia and osteopenia at both 10 and 23 weeks. The biomechanical relevance of these findings was demonstrated by increased bone fragility and ductility in Enpp1 asj/asj mice. In summary, ENPP1 exerts a gene dose effect such that humans with heterozygous ENPP1 deficiency exhibit intermediate levels of plasma analytes associated with bone mineralization disturbance resulting in early onset osteoporosis. © 2019 The Authors. Journal of Bone and Mineral Research published by American Society for Bone and Mineral Research.
Asunto(s)
Raquitismo Hipofosfatémico Familiar , Osteoporosis , Adulto , Animales , Factor-23 de Crecimiento de Fibroblastos , Factores de Crecimiento de Fibroblastos , Humanos , Masculino , Ratones , Osteoporosis/diagnóstico por imagen , Osteoporosis/genética , Fenotipo , Hidrolasas Diéster Fosfóricas/genética , Pirofosfatasas/genéticaRESUMEN
Diseases of ectopic calcification of the vascular wall range from lethal orphan diseases such as generalized arterial calcification of infancy (GACI), to common diseases such as hardening of the arteries associated with aging and calciphylaxis of chronic kidney disease (CKD). GACI is a lethal orphan disease in which infants calcify the internal elastic lamina of their medium and large arteries and expire of cardiac failure as neonates, while calciphylaxis of CKD is a ubiquitous vascular calcification in patients with renal failure. Both disorders are characterized by vascular Mönckeburg's sclerosis accompanied by decreased concentrations of plasma inorganic pyrophosphate (PPi). Here we demonstrate that subcutaneous administration of an ENPP1-Fc fusion protein prevents the mortality, vascular calcifications and sequela of disease in animal models of GACI, and is accompanied by a complete clinical and biomarker response. Our findings have implications for the treatment of rare and common diseases of ectopic vascular calcification.